Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains

Aims:  The identification of a new compound active against Agrobacterium tumefaciens .
Methods and Results:  The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml⁻¹) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l⁻¹ glucose, 15 g l⁻¹ K₂HPO₄ and 5 g l⁻¹ MgSO₄ 7H₂O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato.
Conclusion:  The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens .
Significance and Impact of the Study:  The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.     

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.     

Genetic transformation of the actinorhizal tree Allocasuarina verticillata by Agrobacterium tumefaciens

We have developed an efficient transformation system for the tropical actinorhizal tree Allocasuarina verticillata using Agrobacterium tumefaciens mediated gene transfer. Mature zygotic embryos were inoculated with the disarmed strain C58C1 carrying, in the binary vector BIN19, the nptll gene, providing kanamycin resistance as a selectable marker, and the reporter gene β-glucuronidase containing an intron. The transformed embryos were cultivated on nutrient medium supplemented with 0.5 µM NAA, 2.5 µM BA, 100 mg l⁻¹ kanamycin and 250 mg l⁻¹ cefotaxime. After 2 months, a 21% transformation frequency was obtained. Within 6–9 months, transgenic plants were recovered from 70% of the transformed calli. The presence of the transgenes was demonstrated by PCR analysis and by the expression of the β-glucuronidase; integration of the T-DNA was confirmed by Southern hybridization. More than 100 transgenic plants from a total of 23 independent transformation events have been successfully established in soil. The possibility to obtain nitrogen-fixing nodules after inoculation of transgenic A. verticillata plants by the actinomycetal strain of Frankia Allo2 was established.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.     

潮霉素在大麦遗传转化中的应用研究

以‘B1202’、‘U202’和‘云引1号’3个大麦品种(系)为试材,研究了潮霉素对大麦愈伤组织生长、分化及成熟种子的影响。结果表明:潮霉素对抗性愈伤的适宜筛选浓度为60 mg/L,分化阶段的适宜筛选浓度为40 mg/L,对转基因后代种子筛选以80 mg/L较为适宜,而不同材料对潮霉素的敏感性存在一定程度的差异。通过PCR检测采用农杆菌介导法转化获得的T0和T1代潮霉素抗性苗,结果证实在大麦遗传转化中采用潮霉素进行筛选是可行的。     

Additional virulence genes and sonication enhance <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated loblolly pine transformation

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.     

Effects of antibiotics on the elimination of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) zygotic embryo explants and on transgenic plant regeneration

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine ( Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase ( hpt) controlled by the cauliflower mosaic virus 35S promoter and terminator, and the uidA reporter gene (GUS) driven by the cauliflower mosaic virus 35S promoter and the terminator of nopaline synthase gene, were used in this study. Exposure to 350 mg l^-1 carbenicillin, claforan, and timentin respectively for up to 6 weeks did not eliminate the Agrobacterium, while antibiotics at 500 mg l^-1 eradicated them from the co-cultivated zygotic embryos. All three antibiotics increased callus growth and shoot regeneration at 350 and 500 mg l^-1 each, but reduced callus growth and shoot regeneration at 650 mg l^-1 when compared with controls. Putative transgenic calli were selected for continued proliferation and differentiation on 4.5 mg l^-1 hygromycin-containing medium. Transformed calli and transgenic plants produced on a selection medium containing 4.5 mg l^-1 hygromycin were confirmed by GUS histochemical assays, by polymerase chain reaction (PCR), and by Southern blot analysis. These results are useful for future studies on optimizing genetic transformation procedures in loblolly pine.     

Effect of acute and chronic ammonia and nitrite exposure on oxygen consumption and growth of juvenile big bellied seahorse

Juvenile big bellied seahorse Hippocampus abdominalis were exposed acutely and chronically to elevated ammonia and nitrite {24 h exposure: 0·01, 5·0, 10·1, 14·8 and 19·9 mg l⁻¹ total ammonia-nitrogen [TA-N] and <0·001, 74·4, 99·2 and 123·6 mg l⁻¹ [NO₂-N] nitrite-nitrogen and 35 days exposure: 0·11, 0·55, 1·67 and 3·07 mg l⁻¹ TAN and <0·001, 0·92, 4·67 and 9·10 mg NO₂-N l⁻¹}. Significant ( P <0·001) increases in oxygen consumption rate and ventilation frequency occurred at 14·8, 19·9 mg l⁻¹ TA-N and 99·2, 123·6 mg l⁻¹ NO₂-N for acutely exposed fish. Oxygen consumption rate was significantly ( P <0·05) elevated at 1·67 and 3·07 mg l⁻¹ TA-N in chronically treated fish and ventilation frequency increased significantly ( P <0·05) at 0·55, 1·67, 3·07 mg l⁻¹ TA-N and 4·59, 9·10 mg l⁻¹ NO₂-N. There were no significant differences in growth between controls and ammonia exposed fish. Mortalities occurred at 14·8, 19·9 mg l⁻¹ TA-N.     

Aluminum borate whisker-mediated production of transgenic tobacco plants

An aluminum borate whiskers-mediated transformation system for calluses of tobacco (Nicotiana tabacum, cv. SR-1) has been developed. A total of 50 small pieces of calluses were vigorously agitated in a liquid medium containing aluminum borate whiskers, pBI221 plasmid carrying the -glucuronidase (GUS) gene, and pBI222 plasmid carrying the hygromycin phosphotransferase (HPT) gene. After treatment, calluses were cultured to select for hygromycin resistance, and three resistant calluses were obtained. Adventitious shoots were produced from each hygromycin-resistant callus and were transferred to rooting medium. A total of three plantlets obtained from each hygromycin-resistant callus were acclimatized and established in soil. Polymerase chain reaction analysis revealed that all the plantlets were cotransformed with both the GUS and HPT genes. Detached leaves of transgenic individuals showed clear hygromycin resistance when cultured in liquid medium. Histochemical assay for GUS revealed that one of these transgenic plants expressed the GUS gene, indicating coexpression of foreign genes.     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

铁棍山药微型块茎遗传转化体系的建立

怀山药(Dioscorea opposita)遗传转化是对其进行基因功能分析和遗传改良的基础, 但目前国内外尚未见相关报道。以怀山药优良品种铁棍山药(D. opposita cv. ‘Tiegun’)的微型块茎为受体材料, 对影响遗传转化的因素进行优化, 建立了由根癌农杆菌介导的山药遗传转化体系。过表达质粒载体pCAMBIA1301-DoSERK2含GUS标记基因和潮霉素(Hyg)抗性筛选基因, 沉默质粒载体pART27-DoSERK2含卡那霉素(Kan)抗性筛选基因。根癌农杆菌抑制剂特美汀(Tim)的最佳浓度为500 mg·L ^-1; 再生芽和生根时, Hyg的最佳浓度分别为15和20 mg·L ^-1, Kan的最佳浓度分别为120和160 mg·L ^-1。对转化植株进行PCR和GUS组织化学检测, 结果显示外源基因已整合到铁棍山药转基因株系的基因组中并在细胞中表达。该研究建立了一套取材便利的铁棍山药遗传转化方法, 对其它品种山药的转化也具有参考价值。     

离子注入水稻愈伤组织提高农杆菌转化效率的初步研究

通过ＧＵＳ报告基因表达的组织化学染色分析,我们发现低能氮离子束注入水稻愈伤组织可显著提高根癌农杆菌介转基因效率,这为克服单子叶植物对根癌农杆菌不敏感性、提高遗传转化效率的研究提供了一条新的途径。     

Morphological changes in the gills of Lophiosilurus alexandri exposed to un-ionized ammonia

The average lethal concentration of un-ionized ammonia (48-h LC₅₀NH₃) has been determined by the static assay for larvae (0.48 mg l⁻¹) and alevins (0.92 mg l⁻¹) of 'pacamã' Lophiosilurus alexandri. Studies by light and scanning electron microscopes at the greatest concentration of NH₃ (0.99 mg l⁻¹ for larvae and 1.5 mg l⁻¹ for alevins) have shown that the changes in the cells and branchial tissue were more intense in the alevins.     

Chronic toxicity of ammonia to juvenile gilthead seabream Sparus aurata and related histopathological effects

Effect of 20 days exposure of juvenile gilthead seabream ( Sparus aurata ) to elevated levels of ammonia on growth and survival was examined in a continuous flow system. Suppressed growth and reduced survival were observed at concentrations of 8.2 and 13 mg l⁻¹ total ammonia-N (0.5 and 0.7 mg l⁻¹ un-ionized ammonia-N, respectively) and higher. The maximum acceptable toxic concentration (MATC) for growth was between 4.8 to 8.2 mg l⁻¹ total ammonia-N (0.3 and 0.5 mg l⁻¹ un-ionized ammonia-N, respectively). Fish exposed to high ammonia levels (13 mg l⁻¹ total ammonia-N, 0.7 mg l⁻¹ un-ionized ammonia-N) displayed clear signs of liver pathology. Existing evidence suggests that S. aurata is less sensitive to ammonia than other reported marine and freshwater fish.
Under certain conditions ammonia concentration in the intensive fish ponds in Eilat may exceed the no observed effect concentration for S. aurata .     

Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes

Abstract Efficient transformation of strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes by electroporation with binary Ti plasmid vector is reported. This procedure yields rates of transformation of 10⁶-10³ per μg DNA, which is several orders of magnitude greater than previously published procedures for this genus, the efficiency of transformation varies with the bacterial strain used. This procedure will be useful for the construction of plant DNA libraries directly in Agrobacterium .     

Assessment of microbial involvement in the elevation of copper levels in drinking water

A study of bacterial populations in metropolitan Adelaide domestic reticulation pipes was conducted to investigate a possible link between copper in drinking water and biofilms. Biofilm densities from cold water copper pipes at 10 sample sites were measured by viable cell counts. The range detected was from <2 × 10¹ to 3·25 × 10⁷ cfu cm⁻². Five isolates were selected for further experiments as they represented a range of responses to solvated copper and relative tendency for adhesion on glass slides. Drinking water supplied to the Adelaide Hills is high in total organic carbon (TOC; 22·57 mg C l⁻¹) and has a negative Langelier Index (LI; −1·16), whereas Adelaide metropolitan water undergoes filtration and has both a lower TOC and LI (10·72 mg C l⁻¹, LI, −0·49). Copper coupons were exposed to biofilm isolates (24 h), washed and resuspended in Adelaide metropolitan and Adelaide Hills water. Copper coupons not exposed to biofilm isolates were suspended in respective waters as a control. After 5 d of incubation, the copper content of Adelaide Hills water (4·71 ± 0·87 mg Cu l⁻¹), in which the copper coupons were suspended, consistently exceeded values obtained in the metropolitan Adelaide water (1·17 ± 0·249 mg Cu l⁻¹). The concentration of copper in the Adelaide Hills water was influenced by the bacterial species forming the biofilm on the coupon, with Agrobacterium sp. producing significantly higher levels of soluble copper than the control. The experiments reported here indicate that the suspended organic carbon, the aggressivity of the water and the biofilm may independently or synergistically increase the dissolution of copper from pipes into drinking water.     

The effects of variable nitrogen and phosphorous concentrations on Eriophorum vaginatum tillers grown in nutrient solutions

Eriophorum vaginatum tillers were collected at Eagle Creek, Alaska and cultivated in aerated solutions under controlled environmental conditions. The nutrient solutions contained traces, 1.05 and 21 mg l⁻¹ N (nitrate) and traces, 0.15 and 3.10 mg l⁻¹ P (phosphate), pH was maintained at 5.5. The high N, 21 mg l⁻¹, and P, 3.18 mg l⁻¹, nutrient solution produced significant biomass increases. Functional leaf areas were significantly enhanced by high N and P doses in the solutions. Root surface areas varied considerably between treatments; however, significant differences were not found. The mean root surface area of a tiller reached 126 cm² (range 35–290 cm²), whereas the functional leaf area was 6.8 cm² (range 3.3–20.3 cm²). Tillers growing in the highest N + P solutions produced twice the number of daughter tillers as tillers growing in solutions with trace amounts of N and P.     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

Genetic transformation ofPelargonium X hortorum

Summary TransgenicPelargonium X hortorum have been producedvia Agrobacterium tumefaciens-mediated transformation. The regeneration protocol used provided a regeneration frequency approximately to 95 percent. Clumps of regenerants, from cotyledons and hypocotyls ofPelargonium X hortorum seedlings, were inoculated with the disarmed strain EHA101 ofAgrobacterium tumefaciens. This strain contains a binary vector carrying neomycin phosphotransferase II, hygromycin B phosphotransferase and ß-glucuronidase genes. Selection on the regeneration medium supplemented with hygromycin allowed production of transgenic plants in up to 20% of the inoculated explants. The insertion of foreign DNA was demonstrated by Southern and polymerase chain reaction analysis: these experiments indicated that the inserted T-DNA is not full length for most of the plants. All RO transgenic plants exhibited a normal phenotype and are fertile.Abbreviations GUS ß-glucuronidase coding sequence - PCR polymerase chain reaction - CaMV cauliflower mosaic virus - NPTII neomycin phosphotransferase coding sequence - NOS nopaline synthase gene promoter and terminator - HPH hygromycin B phosphotransferase coding sequence - SDS sodium dodecyl sulphate - EDTA (ethylenedinitro trilo)tetra-acetic acid disodium salt